The objective of this work is to use bound enzymes for the total hydrolysis and sequencing of polypeptides under conditions which do not modify side chains. Specifically our goals include: 1) total hydrolysis with mixtures of proteolytic enzymes bound to Glycophase-G, a glyceryl derivative of porous glass. This support material for the enzymes should eliminate non-specific adsorption and thus permit digestion of small amounts of polypeptide; 2) sequencing of the C-terminus with a collection of bound carboxypeptidases of different specificity. An ultrafilter reactor would allow convenient separation of amino acids from polypeptide and easy removal of polypeptide from enzyme; 3) we propose a new isolation and immobilization of the enzyme pyrrolidonecarboxylpeptidase which removes the pyrrolidonecarboxyl group (cyclyzed glutaminyl) from N-termini of polypeptides. A stable, immobilized derivative of the enzyme would be very useful in conventional sequencing approaches. The use of bound enzymes rather than soluble enzymes is based on the following facts: 1) bound enzymes cannot digest themselves or other bound enzymes; 2) bound enzymes are reusable; 3) they are more stable; 4) they may be conveniently separated from the reaction mixture; and 5) processes based on immobilized enzymes are readily automatable. BIBLIOGRAPHIC REFERENCES: Royer, G. P., Liberatore, F. A., Liberatore, F. A., and Schwartz, W., Methods in Enzymol. 47, 40 (1977). Liberatore, F. A., McIsaac, J. E., and Royer, G. P., FEBS Letters 68, 45 (1976).